Product formation and kinetic simulations in the pH range 1-14 account for a free-radical mechanism of peroxynitrite decomposition

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).     

Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 +/- 3.9% (mean +/- SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 +/- 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 +/- 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 +/- 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.     

Nitric oxide detection and visualization in biological systems. Applications of the FNOCT method

Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) were applied to specifically trap nitric oxide (NO) with high sensitivity. The fluorescent o-quinoid pi-electron system of the FNOCTs (lambda(exc) = 460 nm, lambda(em) = 600 nm) reacts rapidly with NO to a fluorescent phenanthrene system (lambda(exc) = 380 nm, lambda(em) = 460 nm). The cyclic nitroxides thus formed react further to non-radical products which exhibit identical fluorescence properties. Using the acid form of the trap (FNOCT-4), NO release by spermine NONOate and by lipopolysaccharide (LPS)-activated alveolar macrophages were studied. A maximum extracellular release of NO of 37.5 nmol h(-1) (10(6) cells)(-1) from the macrophages was determined at 11 h after activation. Furthermore, intracellular NO release by LPS-activated macrophages and by microvascular omentum endothelial cells stimulated by the Ca2+ ionophore A-23187, respectively, was monitored on the single cell level by means of fluorescence microscopy. After loading the cells with the membrane-permeating acetoxymethylester derivative FNOCT-5, which is hydrolyzed to a non-permeating dicarboxylate by intracellular hydrolases, NO formation by the endothelial cells started immediately upon stimulation, whereas start of NO production by the macrophages was delayed with a variation between 4 and 8 h for individual cells. These results demonstrate that the FNOCTs can be used to monitor NO release from single cells, as well as from NO-donating compounds, with high sensitivity and with temporal and spatial resolution.     

Dose-dependent effects of endotoxin on human sleep

The role of the central nervous system in the host response to infection and inflammation and modulation of these responses by the hypothalamic-pituitary-adrenal system are well established. In animals, activation of host defense mechanisms increases non-rapid eye movement (NREM) sleep amount and intensity, which, in turn, are thought to support host defense, or the body's ability to defend itself against challenges to its immune system. In humans, the evidence is conflicting. Therefore, we investigated the effects of three placebo-controlled doses of endotoxin on host response, including nocturnal sleep in healthy volunteers. Administered before nocturnal sleep onset, endotoxin dose dependently increased rectal temperature, heart rate, and the plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-1 receptor antagonist, IL-6, and cortisol. The lowest dose reliably increased circulating levels of cytokines and soluble cytokine receptors, but it did not affect rectal temperature, heart rate, or cortisol. This subtle host defense activation increased deep NREM sleep amount, often referred to as slow-wave sleep (stages 3 and 4), and intensity (delta power). Conversely, the highest dose of endotoxin disrupted sleep. Whereas it is well established that the endocrine and thermoregulatory systems are very sensitive to endotoxin, this study shows that human sleep-wake behavior is even more sensitive to activation of host defense mechanisms.     

BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy

Weinvestigated the influence of pregnancy on large-conductancecalcium-activated potassium channel (BK_Ca) activity(NP_o) and on channel expression in membranes ofisolated human myometrial smooth muscle cells.NP_o in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and thehalf-maximal activation potential was shifted by 39 mV to more negativepotentials. This effect was not due to an enhanced BK_Cachannel expression. In the presence of cAMP kinase (PKA) or cGMP kinase(PKG), NP_o increased in patches from PMbut decreased in those from NPM. Western blot analysis and use of aspecific PKG inhibitor (1 µM KT-5823) verified the existence of apartially active membrane-associated PKG. Inhibition of PKA by100 nM PKI, the inhibitory peptide of PKA, had no effect onNP_o. 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect wasabolished by iberiotoxin, a specific blocker of BK_Cachannels. It is concluded that an endogenous, membrane-bound PKG inmyometrial cells specifically enhances BK_Ca channelactivity during pregnancy and thus may contribute to uterine quiescenceduring pregnancy.

     

The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism

We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58(IPK), the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2alpha. P58(IPK) also inhibits PERK, an eIF2alpha kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58(IPK) to interact with and inhibit multiple eIF2alpha kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58(IPK) during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58(IPK-/-) mice, we demonstrated that the absence of P58(IPK) led to an increase in eIF2alpha phosphorylation and decreased influenza virus mRNA translation. The absence of P58(IPK) also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58(IPK) target, PKR, the trends were reversed-eIF2alpha phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58(IPK) also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2alpha phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2alpha. Taken together, our results support a model in which P58(IPK) regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.     

Novel technique shows different hydrophobic chemical signatures of exotic and indigenous plant soils with similar effects of extracts on indigenous species seedling growth

Changes to ecosystem abiotic parameters are regarded as possible mechanisms facilitating plant invasion and community composition shifts. This study compared the hydrophobic chemical signatures of soil from exotic bitou bush (Chrysanthemoides monilifera spp. rotundata) invaded, indigenous acacia (Acacia longifolia var. sophorae) dominated and bare sand (unvegetated) habitats using a novel, rapid, capturing technique which utilised Amberlite® XAD4 resin filled bags that were placed in situ. The hydrophobic chemical signature of the bitou bush soil extract was significantly different to the acacia soil and bare sand extracts. High concentrations of 18 sesquiterpenes dominated the hydrophobic signature of the bitou bush extract. Low concentrations of all three extracts did not significantly affect the seedling growth of three indigenous test species under laboratory conditions, however, at higher concentrations, the extracts from soil inhabited by plants, whether exotic or indigenous, similarly inhibited the seedling growth of two species, while seedling growth of the third species was inhibited by extracts from all three soil types. These results do not support the hypothesis that exotic invasive species are more likely to exhibit allelopathic effects than indigenous plant species.